Synthesis of novel inhibitors of isoprenoid biosynthesis (ISPF)

ABSTRACT

Antibacterial and antimalarial IspF inhibitor compounds and compositions are described. Methods include administering described compounds and compositions to treat bacterial or parasitic infections and to inhibit parasite or bacterial growth.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of co-pending U.S. application Ser. No.14/390,548, filed Oct. 3, 2014, which is a U.S. nationalization under 35U.S.C. §371 of International Application No. PCT/US2013/037765, filedApr. 23, 2013, which claims the benefit of priority under 35 U.S.C.§119(e) to U.S. provisional application No. 61/637,642, filed Apr. 24,2012, 61/701,269 filed Sep. 14, 2012 and 61/703,145, filed Sep. 19,2012. The disclosures of the referenced applications are incorporatedherein by reference in their entireties.

BACKGROUND

The fight against malaria, tuberculosis, and other infectious disease isgrowing more difficult due to the emergence of drug resistant forms ofthese diseases. One strategy to address the resistance problem is todevelop novel anti-infective agents that employ new mechanisms ofaction. In recent studies, it has been shown that bacterial andparasitic organisms, such as those involved in malaria, tuberculosis,and melioidosis, use the methylerythritol isoprenoid (MEP) biosyntheticpathway, to produce isoprenoids, which are the basic building blocks ofmany essential substances found in plants and animals. Fortunately,humans do not use this process, which means any foreign pathogen in thehuman body that uses the MEP pathway can be targeted due to thedifferent enzymes it puts to use.

SUMMARY

Materials and methods to design and create different analogs ofoxdihydropyrimidine carboxylic acid, 4-(1H-imidazol-1-yl)phenol and4-(1H-imidazol-5-yl)phenol that inhibit the2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MECDP) synthase, or IspF,enzymes of the MEP pathway, are described. In completing theseobjectives, the use of docking software (SYBYL) was used for thetheoretical design of compounds.

Compounds were designed to inhibit the enzyme IspF present in the MEPpathway. The MEP pathway is a non-mevalonate pathway or2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphatepathway (MEP/DOXP pathway) of isoprenoid biosynthesis. Analogs from theclaimed chemical series were found to inhibit the enzyme IspF fromBurkholderia pseudomallei using SPR and NMR assays at micromolarconcentrations. Selected compounds were shown to have anti-bacterialactivity in Burkholderia thailandensis, a non-fermenting motilegram-negative bacteria that is often used as a model for Burkholderiapseudomallei, a BSL-3 pathogen. Burkholderia pseudomallei is a class Bpathogen and is considered a biosecurity threat. Selected analogs werescreened against Burkolderia pseudomallei and found to haveanti-bacterial activity.

Anti-infective 2-amino-1,6-dihydro-6-oxo-5-pyrimidinecarboxylic acidderivatives are disclosed. The anti-infective2-amino-1,6-dihydro-6-oxo-5-pyrimidinecarboxylic acid derivatives areadministered to a subject, such as a mammal, such as a human or a horse,to treat a bacterial infection. Compounds and compositions as disclosedherein are administered to a subject to inhibit bacterial growth, suchas Burkholderia spp. or Mycobacterium spp., and to treat subjectsinfected by such bacteria. The compounds disclosed in this applicationinhibit the enzymes IspD and or IspF and will be useful for thetreatment of microbial infections. Compounds in this application havedemonstrated anti-bacterial activity against Burkholderia psuedomallei,Burkholderia thailandenesis and Plasmodium falciparum.

Embodiments include compounds of Formula I:

wherein

X is N and Y is C, or X is C and Y is N;

R¹ is OH or OR⁵, wherein R⁵ is lower alkyl, acyl, CH₂OCOCH₃, or aprodrug;

R² is alkyl, aryl, alkylaryl, arylheteroaryl, or heteroaryl;

R³ is H or lower alkyl; and

R⁴ is H or lower alkyl.

Embodiments include compounds of Formula II:

wherein

R¹ is H or lower alkyl;

R² is H or lower alkyl; and

R³ is alkyl, aryl, alkylaryl, arylheteroaryl, or heteroaryl.

Embodiments include compounds of Formula III:

wherein

R¹ is H or lower alkyl;

R² is H or lower alkyl; and

R³ is alkyl, aryl, alkylaryl, arylheteroaryl, or heteroaryl.

The most potent anti-bacterial compound, against Burkholderia in initialtests was HGN-0118 from the pyrimidine series with 94.5% inhibition at 1mM.

The most potent antimalarial compound in initial tests was HGN-0049,submicromolar for all three strains of drug resistant malaria. Anotherpotent compound was HGN-0051.

Another potent antibacterial compound against Burkholderia is from theimidazole series, HGN-0095 with 88.3% inhibition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph depicting B. pseudomallei growth rates (as measuredby absorbance at OD₆₀₀) over time (min) in the presence of IspFinhibitor compounds HGN-0005, -0013, -0015, -0017, -0020, -0023, -0024,-0025, -0026, -0027, -0028, -0029, -0030, -0031, and -0032, and anegative control of no inhibitor.

FIG. 2 shows a graph depicting B. pseudomallei growth rates (as measuredby absorbance at OD₆₀₀)) over time (min) in the presence of IspFinhibitor compounds HGN-0023, -0027, -0028, and -0029.

FIG. 3 shows a graph depicting B. pseudomallei growth rates (as measuredby absorbance at OD₆₀₀) over time (min) in the presence of the IspFinhibitor compound HGN-0023 at concentrations of 1 mM, 500 μM, 250 μM,125 and 62.5 μM; Controls included (a) DMSO and medium without bacteria(the blank); (b) DMSO, and (c) 300 μg/ml of kanamycin.

FIG. 4 shows a graph depicting B. pseudomallei growth rates (as measuredby absorbance at OD₆₀₀) over time (min) in the presence of the IspFinhibitor compound HGN-0027 at concentrations of 1 mM, 500 μM, 250 μM,125 μM, and 62.5 μM. Controls included (a) DMSO and medium withoutbacteria (the blank); (b) DMSO, and c) 300 μg/ml of kanamycin.

FIG. 5 shows a graph depicting B. pseudomallei growth rates (as measuredby absorbance at OD₆₀₀) over time (min) in the presence of the IspFinhibitor compound HGN-0028 at concentrations of 1 mM, 500 μM, 250 μM,125 μM, and 62.5 μM. Controls included (a) DMSO and medium withoutbacteria (the blank); (b) DMSO, and (c) 300 μg/ml of kanamycin.

FIG. 6 shows a graph depicting B. pseudomallei growth rates (as measuredby absorbance at OD₆₀₀) over time (min) in the presence of the IspFinhibitor compound HGN-0029 at concentrations of 1 mM, 500 μM, 250 μM,125 μM, and 62.5 μM. Controls included (a) DMSO and medium withoutbacteria (the blank); (b) DMSO, and (c) 300 μg/ml of kanamycin.

FIG. 7 shows a graph depicting B. pseudomallei strain 1026b growth rates(as measured by absorbance at OD₆₀₀) over time (min) in the presence ofthe IspF inhibitor compound HGN-0027 at concentrations of 1 mM, 500 μM,250 μM, 125 μM, 62.5 μM, and 31.25 μM. Controls included (a) DMSO andmedium without bacteria (the blank); (b) DMSO, and (c) 300 μg/ml ofkanamycin.

FIG. 8 shows a graph depicting B. pseudomallei strain 340 growth rates(as measured by absorbance at OD₆₀₀) over time (min) in the presence ofthe IspF inhibitor compound HGN-0027 at concentrations of 1 mM, 500 μM,250 μM, 125 μM, 62.5 μM, and 31.25 μM. Controls included (a) DMSO andmedium without bacteria (the blank); (b) DMSO, and (c) 300 μg/ml ofkanamycin.

FIG. 9 shows a graph depicting B. pseudomallei strain 1026b growth rates(as measured by absorbance at OD₆₀₀) over time (min) in the presence ofthe IspF inhibitor compound HGN-0028 at concentrations of 1 mM, 500 μM,250 μM, 125 μM, 62.5 μM, and 31.25 μM. Controls included (a) DMSO andmedium without bacteria (the blank); (b) DMSO, and (c) 300 μg/ml ofkanamycin.

FIG. 10 shows a graph depicting B. pseudomallei strain 340 growth rates(as measured by absorbance at OD₆₀₀) over time (min) in the presence ofthe IspF inhibitor compound HGN-0028 at concentrations of 1 mM, 500 μM,250 μM, 125 μM, 62.5 μM, and 31.25 μM. Controls included (a) DMSO andmedium without bacteria (the blank); (b) DMSO, and (c) 300 μg/ml ofkanamycin.

FIG. 11 shows a schematic of isoprenoid metabolism through thenon-mevalonate pathway. The non-mevalonate pathway is the exclusiveroute to isoprenoid biosynthesis in eubacteria and the malaria parasite,P. falciparum. This pathway generates the basic isoprenoid buildingblocks IPP and DMAPP, which are elaborated to create diverse downstreamproducts. Enzyme names are in bold.

DETAILED DESCRIPTION

Antimicrobial drug resistance is an urgent problem in the control andtreatment of many of the world's most serious infections, includingPlasmodium falciparum malaria, tuberculosis, and healthcare associatedinfections with Gram-negative bacteria. Because the nonmevalonatepathway of isoprenoid biosynthesis is essential in eubacteria and P.falciparum and this pathway is not present in humans, there is greatinterest in targeting the enzymes of non-mevalonate metabolism forantibacterial and antiparasitic drug development.

Isoprenoids include a large, diverse group of intracellular metaboliteswith multiple cellular functions, including roles in membrane structure,cellular respiration, and cell signaling [Zhang et al. (2011);Gershenzon and Dudareva (2007)]. There are two distinct biosyntheticroutes for producing isopentenyl diphosphate (IPP) and dimethylallyldiphosphate (DMAPP), the basic isoprenoid building blocks. Mammals,including humans, exclusively utilize the classic metabolic routethrough mevalonate. In contrast, eubacteria, cyanobacteria, and plantchloroplasts use an alternative route that produces the key intermediate1-D-deoxyxylulose 5-phosphate (DOXP) from pyruvate (FIG. 11) (Hunter,2007). In bacteria that use this pathway, including Escherichia coli,Burkholderia pseudomallei and Mycobacterium tuberculosis, geneticstudies have demonstrated that non-mevalonate isoprenoid biosynthesis isessential.

The malaria parasite Plasmodium falciparum annually kills nearly 1million people, primarily young children. There is widespread resistanceto older antimalarial drugs, such as chloroquine, and emergingresistance to new artemisinin-based therapies. P. falciparum alsoutilizes non-mevalonate isoprenoid biosynthesis, which is essential tothe parasite. Novel non-mevalonate pathway inhibitors therefore holdgreat promise as antimicrobial agents with broad activity against majorhuman pathogens, including P. falciparum, B. pseudomallei, Mtuberculosis, and enterobacteria, for which global drug resistance hascreated an urgent need.

Compounds

The development of multi-drug resistance in bacteria and parasites thatcause human disease is a growing problem in world health. The claimedcompounds inhibit a unique enzyme pathway that is only found in certaintypes of infectious disease agents, but not in human beings. Compoundsfrom this series will lead to improved drugs and other treatments forinfectious diseases such as malaria and a range of bacterial infections.Members have shown antibacterial activity against Burkholderiathailandensis.

Materials and methods to design and create different analogs ofoxdihydropyrimidine carboxylic acid that inhibit the IspF enzymes of theMEP pathway are described. Embodiments include substituted pyrimidineand imidazole derivatives, compositions containing the pyrimidine andimidazole derivatives, and methods of treatment using therapeuticallyeffective amounts of the pyrimidine and imidazole derivatives. Compoundsand compositions disclosed herein can be used to treat bacterialdisease, wherein the bacteria utilitze the non-mevalonate pathway, suchas Burkholderia spp. and Mycobacterium spp.

Embodiments include compounds of Formula I:

wherein

X is N and Y is C, or X is C and Y is N;

R¹ is OH or OR⁵, wherein R⁵ is lower alkyl, acyl, CH₂OCOCH₃, or prodrugsfor phenolic groups;

R² is alkyl, aryl, alkylaryl, arylheteroaryl, or heteroaryl;

R³ is H or lower alkyl; and

R⁴ is H or lower alkyl.

Embodiments include compounds of Formula II:

wherein

R¹ is H or lower alkyl;

R² is H or lower alkyl; and

R³ is alkyl, aryl, alkylaryl, arylheteroaryl, or heteroaryl.

Embodiments also include compounds of Formula III:

wherein

R¹ is H or lower alkyl;

R² is H or lower alkyl; and

R³ is alkyl, aryl, alkylaryl, arylheteroaryl, or heteroaryl

Anti-infective 2-amino-1,6-dihydro-6-oxo-5-pyrimidinecarboxylic acidderivatives are disclosed.

Specific compounds are disclosed in Tables 2-5 of Example 37 and Tables6-9 of Example 38 herein.

Pharmaceutical Compositions

Although it is possible for compounds to be administered alone in a unitdosage form, compounds are typically administered in admixture with acarrier as a pharmaceutical composition to provide a unit dosage form. Apharmaceutical composition comprises a pharmaceutically acceptablecarrier in combination with a compound disclosed herein or apharmaceutically acceptable salt thereof.

A pharmaceutically acceptable carrier includes, but is not limited to,physiological saline, ringers, phosphate-buffered saline, and othercarriers known in the art. Pharmaceutical compositions can also includeadditives such as, for example, stabilizers, antioxidants, colorants,excipients, binders, thickeners, dispersing agents, readsorpotionenhancers, buffers, surfactants, preservatives, emulsifiers, isotonizingagents, and diluents. Pharmaceutically acceptable carriers and additivesare chosen such that side effects from the pharmaceutical compound areminimized and the performance of the compound is not canceled orinhibited to such an extent that treatment is ineffective.

Methods of preparing pharmaceutical compositions containing apharmaceutically acceptable carrier in combination with a therapeuticcompound or a pharmaceutically acceptable acid addition salt of acompound are known to those of skill in the art. The invention alsoincludes pharmaceutically acceptable salts of the compounds of theinvention. The compounds of the invention are capable of forming bothpharmaceutically acceptable acid addition and/or base salts.

All methods can include the step of bringing one of the compoundsdisclosed herein in association with a carrier and one or moreadditives. Formulations generally are prepared by uniformly andintimately bringing a compound into association with a liquid carrier ora finely divided solid carrier or both, and then, if necessary, shapingthe product into the desired unit dosage forms.

As used herein, the term “pharmaceutically acceptable carrier” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionthat, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform may be accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations may also be prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissues.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid; b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,acetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. They mayoptionally contain opacifying agents and can also be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions that can be used include polymericsubstances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches.

Compositions may also be formulated for delivery as a liquid aerosol orinhalable dry powder.

The compounds may also be formulated for use as topical powders andsprays that can contain, in addition to the compounds of this invention,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body.

According to the methods of treatment of the present invention,bacterial infections are treated or prevented in a patient such as ahuman or lower mammal by administering to the patient a therapeuticallyeffective amount of a compound of the invention, in such amounts and forsuch time as is necessary to achieve the desired result. By a“therapeutically effective amount” of a compound of the invention ismeant a sufficient amount of the compound to treat bacterial orparasitic infections, at a reasonable benefit/risk ratio applicable toany medical treatment. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular patient will depend upon a variety of factors includingthe disorder being treated and the severity of the disorder, theactivity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

A “kit” used in the instant application can include a containercomprising the pharmaceutical compositions and may also include dividedcontainers such as a divided bottle or a divided foil packet. Thecontainer can be in any conventional shape or form as known in the artthat is made of a pharmaceutically acceptable material, for example apaper or cardboard box, a glass or plastic bottle or jar, a resealablebag (for example, to hold a “refill” of tablets for placement into adifferent container), or a blister pack with individual doses forpressing out of the pack according to a therapeutic schedule. Thecontainer employed can depend on the exact dosage form involved, forexample a conventional cardboard box would not generally be used to holda liquid suspension. It is feasible that more than one container can beused together in a single package to market a single dosage form. Forexample, tablets may be contained in a bottle that is in turn containedwithin a box.

A “daily dose” can be a single tablet or capsule or several tablets orcapsules to be taken on a given day.

The kits of the present invention may also include, in addition to ISPFinhibitors, one or more additional pharmaceutically active compounds.Preferably, the additional compound is another ISPF inhibitor or anothercompound useful to treat bacterial or parasitic infections. Theadditional compounds may be administered in the same dosage form as theISPF inhibitor compound or in different dosage forms. Likewise, theadditional compounds can be administered at the same time as the ISPFinhibitor compound(s) or at different times.

Methods of Treatment

Burkholderia spp. cause a number of diseases. For instance, Burkholderiamallei is the etiologic agent of glanders. While glanders primarilyaffects horses, humans, dogs, cats, goats, mules, and donkeys can alsocontract glanders. Glanders often manifests itself as pulmonaryinfection. In pulmonary infections, pneumonia, pulmonary abscesses, andpleural effusion can occur. Glanders can also be a localized infectionof open wounds and of mucus membranes in the eyes, nose, and respiratorytract. Burkholderia thailandensis is an opportunistic pathogen that cancause pneumonia and septicemia. Burkholderia pseudomallei the causativeagent of melioidosis.

In an embodiment, a compound or composition disclosed herein can beadministered to a subject to treat a Burkholderia infection. In anembodiment, compounds and compositions disclosed herein can beadministered to a subject to treat glanders. An embodiment includesadministering a compound or composition disclosed herein to a subjectconcurrently with the administration of at least one of tetracycline,ciprofloxacin, streptomycin, novobiocin, gentamicin, imipenem,ceftrazidime, or a sulfonamide. In another embodiment, compounds andcompositions disclosed herein can be administered to a subject to treatmelioidosis.

Multidrug resistant tuberculosis (MDR-TB), and even extensively drugresistant tuberculosis (XDR-TB) has become more prevalent in the last 20to 40 years. New treatments are sought to battle the rising rates ofdrug resistant TB cases. Mycobacterium tuberculosis, the etiologic agentof tuberculosis, generates isopentenyl pyrophosphate (IPP) anddimethylallyl pyrophosphate (DMAPP) via the non-mevalonate pathway.Thus, M. tuberculosis is a target for the compounds and compositionsdisclosed herein. Treatment with compounds and compositions as disclosedherein represent new treatments for tuberculosis. As such, an embodimentincludes administering a compound or composition as disclosed herein toa subject with tuberculosis. The treatment with the compounds andcompositions disclosed herein may optionally be administered with atleast one of the first line drugs ethambutol, isoniazid, rifampicin, orpyrazinamide. The treatment with the compounds and compositionsdisclosed herein may optionally be administered with at least one of thesecond line drugs an aminoglycoside (e.g., streptomycin, kanamycin,amikacin), a fluoroquinolone (e.g., ciprofloxacin, ofloxacin,sparfloxacin, moxifloxacin, etc.), capreomycin, viomycin, enviomycin, athioamide (e.g., ethionamide and protionamide), cycloserine,para-aminosalicylic acid, thiacetazone, clofazimine, linezolid, amacrolide (e.g., clarithromycin, azithromycin), oramoxicillin/clavulanate.

Methods of treating bacterial or parasitic infection includeadministering a compound or composition as described herein, andoptionally further comprising administering at least one otherantibacterial agent.

Definitions

The term “compound disclosed herein” and similar terms refers to an IspFinhibitor compound as described herein, a compound of formulae I, II, orIII or compounds of Tables 2-9, or a compound described in the Examples,or a pharmaceutically acceptable salt, solvate, clathrate, hydrate,polymorph or prodrug thereof.

Compounds disclosed herein may contain one or more chiral centers and/ordouble bonds and, therefore, exist as stereoisomers, such as double-bondisomers (i.e., geometric isomers), enantiomers, or diastereomers.According to this invention, the chemical structures depicted herein,including the compounds of this invention, encompass all of thecorresponding compounds' enantiomers, diastereomers and geometricisomers, that is, both the stereochemically pure form (e.g.,geometrically pure, enantiomerically pure, or diastereomerically pure)and isomeric mixtures (e.g., enantiomeric, diastereomeric and geometricisomeric mixtures). In some cases, one enantiomer, diastereomer orgeometric isomer will possess superior activity or an improved toxicityor kinetic profile compared to other isomers. In those cases, suchenantiomers, diastereomers and geometric isomers of compounds of thisinvention are preferred.

The term “alkyl” refers to a saturated linear or branched hydrocarbonradical. In one embodiment, alkyl has from 1 to 8 carbon atoms. Inanother embodiment, alkyl has from 1 to 6 carbon atoms. In anotherembodiment, alkyl has from 1 to 4 carbon atoms. In one embodiment, alkylhas 1 carbon. The alkyl group may optionally be substituted with one ormore substituents such as fluorine, chlorine, alkoxy groups having from1 to 8 carbon atoms (e.g., methoxy or ethoxy), or amido groups havingfrom 1 to 8 carbon atoms, such as acetamido. These substituents maythemselves be substituted with one or more functional groups such ashydroxy groups, carboxy groups, acetoxy groups, or halogens.

As used herein “aryl” means a mono- or poly-nuclear aromatic hydrocarbonradical. Examples of “aryl” groups include, but are not limited toaromatic hydrocarbons such as a phenyl group or a naphthyl group. Thearomatic group may optionally be substituted with one or moresubstituents such as fluorine, chlorine, alkyl groups having from 1 to 8carbon atoms (e.g., methyl or ethyl), alkoxy groups having from 1 to 8carbon atoms (e.g., methoxy or ethoxy), alkoxyalkyl groups having from 1to 8 carbon atoms and one or more oxygen atoms, or amido groups havingfrom 1 to 8 carbon atoms, such as acetamido. These substituents maythemselves be substituted with one or more functional groups such ashydroxy groups, carboxy groups, acetoxy groups, or halogens.

In one embodiment, aryl is a phenyl group or a naphthyl group that iseither unsubstituted or substituted.

In another embodiment, aryl is a heteroaryl in which one or more of thecarbon atoms of an aromatic hydrocarbon is substituted with a nitrogen,sulfur, or oxygen. Examples of a “heteroaryl” include, but are notlimited to pyridine, pyrimidine, pyran, dioxin, oxazine, andoxathiazine. Likewise, the heteroaryl may optionally be substituted withfunctional groups such as hydroxy groups, carboxy groups, halogens, andamino groups.

The term “lower” refers to a group having up to four atoms. For example,a “lower alkyl” refers to an alkyl radical having from 1 to 4 carbonatoms, “lower alkoxy” refers to “—O—(C₁-C₄)alkyl. Included in thisdefinition is a lower group having 1 to 3 carbon atoms.

The term “cycloalkyl” refers to a saturated, mono- or polycyclic alkylradical having from 3 to 20 carbon atoms. Representative cycloalkylsinclude cyclopropyl, 1-methylcyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, -cyclodecyl,octahydro-pentalenyl, and the like. Cycloalkyl groups may be optionallysubstituted with one or more substituents.

As used herein, the term “haloalkyl” means an alkyl group, in which oneor more (including all) the hydrogen radicals are replaced by a halogroup, wherein each halo group is independently selected from —F, —Cl,—Br, and —I. The term “halomethyl” means a methyl in which one to threehydrogen radical(s) have been replaced by a halo group. Representativehaloalkyl groups include trifluoromethyl, bromomethyl,1,2-dichloroethyl, 4-iodobutyl, 2-fluoropentyl, and the like.

The term “polymorph” refers to solid crystalline forms of a compound orcomplex thereof. Different polymorphs of the same compound can exhibitdifferent physical, chemical and/or spectroscopic properties. Differentphysical properties include, but are not limited to stability (e.g., toheat or light), compressibility and density (important in formulationand product manufacturing), and dissolution rates (which can affectbioavailability). Differences in stability can result from changes inchemical reactivity (e.g., differential oxidation, such that a dosageform discolors more rapidly when comprised of one polymorph than whencomprised of another polymorph) or mechanical characteristics (e.g.,tablets crumble on storage as a kinetically favored polymorph convertsto thermodynamically more stable polymorph) or both (e.g., tablets ofone polymorph are more susceptible to breakdown at high humidity).Different physical properties of polymorphs can affect their processing.For example, one polymorph might be more likely to form solvates ormight be more difficult to filter or wash free of impurities thananother due to, for example, the shape or size distribution of particlesof it.

The term “substituent” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, alkyl,alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H,perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy,carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)nalkyl (where nis 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n) heteroaryl (where n is0-2), S(O)_(n) heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl,cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester(alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl,heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-,alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstitutedaryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, andunsubstituted cycloalkyl. In one aspect, the substituents on a group areindependently any one single, or any subset of the aforementionedsubstituents.

The term “antibacterial” refers to compounds or compositions disclosedherein that have either bactericidal or bacteriostatic activity. An“antibacterial” compound or composition in this context can inhibit thegrowth of B. mallei, B. pseudomallei, and other Burkholderia spp., andother gram-negative bacteria. Additionally, an antibacterial compound orcomposition in this context can inhibit the growth of Mycobacteriumtuberculosis and other Mycobacterium spp. The term “inhibiting thegrowth” indicates that the rate of increase in the numbers of apopulation of particular bacteria is reduced. Thus, the term includessituations in which the bacterial population increases but at a reducedrate, as well as situations where the growth of the population isstopped, as well as situations where the numbers of the bacteria in thepopulation are reduced or the population even eliminated.

An “antimicrobial agent” refers to a substance that kills microbes orinhibits microbial growth or replication. Microbes are microorganismsthat include bacteria, fungi, or protozoans. An antimicrobial agent canbe an antibiotic (e.g., streptomycin) or can be an non-pharmaceuticalantimicrobial (e.g., chlorhexidine, silver, triclosan).

The terms “treat” and “treatment” refer to therapeutic treatment,wherein the object is to inhibit or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofinfection. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration, or palliation of the disease state. “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the condition or disorder is tobe prevented.

“Mammal” for purposes of treatment or therapy refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, and thelike. Preferably, a mammal is human.

EXAMPLES Example 1 Analyis of IspF

To design inhibitor compounds, IspF was analyzed to better designedcompounds to inhibit its function.

IspF Screening by NMR Spectroscopy

To design IspF inhibitor compounds, the IspF protein was analyzed bynuclear magnetic resonance (NMR) spectroscopy. NMR samples were preparedby diluting tagless, concentrated BpIspF protein in SEC buffer to 20 μm(60 μm monomer) with NMR buffer (10 mM K-Phos (pH 7.8), 50 mM NaCl, 10%(v/v) 2H₂O). Compounds were assayed at ligand concentrations of 400 μm,with 400 μm cytidine and 20 μM deuterated dimethyl sulfoxide (d6-DMSO)present in a 500 μM sample volume. All experiments were conducted on a600-MHz Bruker AV spectrometer with TCI cryoprobe set to 280 K.Screening was done using ligand-observe, protonbased one-dimensionalsaturation transfer difference nuclear magnetic resonance (STD-NMR)(Mayer et al., 1999) and two-dimensional nuclear Overhauser effectspectroscopy (NOESY) according to (Begley et al, 2010).

Briefly, 32 scans and 32,000 points were acquired over a 14 ppm sweepwidth for STD-NMR data, with a total recycle delay of 4.0 s for eachmixture. A low-power 30-ms spin-lock pulse was added to filter outlow-level protein peaks, and a WATERGATE sequence added to suppress bulkwater signal (Piotto et al., 1992). STD-NMR pre-saturation was doneusing a 3.0 s-long train of Gaussian-shaped pulses with a spectral widthof 600 Hz focused at −1.0 ppm, with reference irradiation set to 30 ppm.For NOESY experiments, 2,048 9 160 points were collected with a mixingtime of 500 ms and a recycle delay of 2.0 s, with WATERGATE solventsuppression for each mixture.

Sequence Analysis

The IspF from nine pathogens were aligned and studied for sequenceconservation. The nine pathogens were Escherichia coli, Yersinia pestis,Haemophilus influenzae, Burkholderia pseudomallei, Mycobacteriumtuberculosis, Plasmodium vivax, Brucella melitensis, Babesia bovis, andPlasmodium falciparum (IspF subunit from the bifunctional IspDF enzyme).Of note, D48 was 100% conserved in the 9 pathogens. This amino acidresidue does not bind substrate and has a putative structural role and aputative functional role of assisting Zn²⁺ ion position duringcatalysis. There was also 100% conservation in having the “DIG” motif,whereby D58 binds ribose and G60 is necessary for the conserved pseudoβ-turn.

Additionally screening and substrate-derived fragments were soaked intocrystals of IspF from B. pseudomallei. This allowed for screening ligandbinding and conformation selection as analyzed by the drug designsoftware AutoDocks (version 4.0) (The Scripps Research Institute, LaJolla, Calif.). Through this software, specific candidate compounds andclasses of candidate compounds were selected.

Example 2 Synthesis of Sodium2-((4-chlorophenethyl)amino-4-oxidopyrimidine-5-carboxylate (HGN-0028)

A mixture of ethyl2-((4-chlorophenethyl)amino)-6-oxo-1,6-dihydropyrmidine-5-carboxylate(60 mg, 1.86 mmol) and sodium hydroxide (1M) was heated up to reflux for4 hours and then cooled to 40° C. overnight. After heating, the solutionwas completely dissolved without precipitate and condensed. ¹H-NMRconfirmed the product as sodium2-((4-chlorophenethyl)amino-4-oxidopyrimidine-5-carboxylate (HGN-0028)(59 mg, 94% yield).

Example 3 Synthesis of ethyl6-oxo-2-(((tetrahydrofuran-2-yl)methyl)amino)-1,6-dihydropyrimidine-5-carboxylate(HGN-0017)

(tetrahydrofuran-2-yl)methanamine (142 mg, 1.40 mmol) was added intoethyl 2-(methylthio)-6-oxo-1,6-dihydropyrmidine-5-carboxylate (300 mg,1.40 mmol) in methanol and refluxed overnight. After half of thestarting material was consumed, the reaction mixture was reheated againovernight. Then the reaction mixture was cooled and the precipitatefiltered. ¹H-NMR confirmed the product as ethyl6-oxo-2-(((tetrahydrofuran-2-yl)methyl)amino)-1,6-dihydropyrimidine-5-carboxylate(HGN-0017) (176 mg, 47% yield).

Example 4 Synthesis of ethyl2-((1,1′-biphenyl)-4-ylamino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(HGN-0033)

Ethyl 2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (60 mg,0.233 mmol) was added to 1,1′-biphenyl-4-amine (79 mg, 0.467 mmol)dissolved in ethanol. The solution was heated in an oil bath to 80° C.,while stirring, in reflux, for 72 hours. The sample was filtered anddried. ¹H-NMR confirmed the structure (31 mg, 40% yield).

Example 5 Synthesis of ethyl6-oxo-2-((4-(piperidin-1-yl)phenyl)amino)-1,6-dihydropyrimidine-5-carboxylate(HGN-0034)

Ethyl 2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (200 mg,0.934 mmol) was added to 4-(piperidin-1-yl)aniline (158 mg, 0.934 mmol)dissolved in ethanol. The solution was heated in an oil bath to 80° C.,while stirring, in reflux, for 72 hours. The sample was filtered anddried. ¹H-NMR confirmed the product (HGN-0034) (15 mg, 0.447 mmol).

Example 6 Synthesis of2-((1,1′-biphenyl)-4-ylamino)-6-oxo-1,6-dihydropyrimidine-5-carboxylicacid (HGN-0035)

A mixture of ethyl2-((1,1′-biphenyl)-4-ylamino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(100 mg, 0.298 mmol), sodium hydroxide (1M, aq), and methanol wasstirred at 70° C. for 2 hours and for 40° C. overnight. The ester didnot dissolve in 3 mL methanol. The ester was then put in 3 mL dioxaneand heated to 103° C. for 4 hours with an additional 1 mL of NaOH added.The reaction mixture was then heated at 40° C. overnight. After theovernight heating, the reaction mixture was cooled and the solventremoved. The reaction mixture was diluted with water, and the pH wasadjusted to about 3.0. The reaction mixture was filtered to obtain theend product. ¹H-NMR confirmed the product (55 mg, 60.0% yield).

Example 7 Synthesis of6-oxo-2-((4-(piperidin-1-yl)phenyl)amino)-1,6-dihydropyrimidine-5-carboxylicacid

A mixture of ethyl6-oxo-2-((4-(piperidin-1-yl)phenyl)amino)-1,6-dihydropyrimidine-5-carboxylate(100 mg, 0.298 mmol), sodium hydroxide (1M, aq), and methanol wasstirred at 70° C. for 2 hours and for 40° C. overnight. The reactionmixture was filtered to remove the solid precipitate to verify product.Recombined the filtrate and precipitate to concentrate by adding 3 mLdioxane and heated to 103° C. for 4 hours with an additional 1 mL ofNaOH added. The reaction mixture was then heated at 40° C. overnight.After the overnight heating, the reaction mixture was cooled and thesolvent removed. The reaction mixture was diluted with water, and the pHwas adjusted to about 3.0. The reaction mixture was filtered to obtainthe end product. ¹H-NMR confirmed the product (55 mg, 60% yield).

Example 8 Synthesis of ethyl2-((4-(morpholinophenylamino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(HGN-0037)

Ethyl 2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (311 mg,1.452 mmol) and 4-morpholinoaniline (259 mg, 1.452 mmol) were dissolvedin 10 mL of ethanol and heated at 80° C. overnight. The reaction mixturewas filtered to obtain the precipitate. ¹H-NMR confirmed the product(294 mg, 58.8% yield).

Example 9 Synthesis of ethyl2-((1-(tert-butoxycarbonyl)peperidin-4-yl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate

Ethyl 2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (292 mg,1.365 mmol) and tert-butyl-4-aminopiperidine-1-carboxylate (273 mg,1.365 mmol) were dissolved in 10 mL of ethanol and heated at 80° C.overnight. The precipitate was not evident so the solution was heated to90° C. overnight. The final product was then recovered.

Example 10 Synthesis of ethyl2-((1-benzylpeperidin-4-yl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate

Ethyl 2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (301 mg,1.403 mmol) and 1-benzylpiperidine-4-amine (267 mg, 1.403 mmol) weredissolved in 10 mL of ethanol and heated at 80° C. overnight. Theprecipitate was not evident so the solution was heated to 90° C.overnight. The final product was then recovered.

Example 11 Synthesis of ethyl2-((1-benzylpyrrolidin-3-yl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate

Ethyl 2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (313 mg,1.460 mmol) and 1-benzylpyrrolidin-3-amine (257 mg, 1.460 mmol) weredissolved in 10 mL of ethanol and heated at 80° C. overnight. Theprecipitate was not evident so the solution was heated to 90° C.overnight. The final product was then recovered.

Example 12 Synthesis of Ethyl2-((4-chlorophenethyl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(HGN-0038)

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (333 mg, 1.554mmol) and 2-(4-chlorophenyl)ethanamine (242 mg, 1.554 mmol) weredissolved in 10 mL of ethanol and heated at 80° C. overnight. Afterheating, the solution was run on thin layer chromatography and filtered.The precipitate was collected and stored. ¹H-NMR confirmed the product(126 mg, 25.2% yield).

Example 13 Synthesis of Ethyl2-([1,1′-biphenyl]-3-ylamino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (319 mg, 1.491mmol) and [1,1′-biphenyl]-3-amine (252 mg, 1.491 mmol) were dissolved in10 mL of ethanol and heated at 80° C. overnight. After heating, thesolution was run on thin layer chromatography and filtered. Theprecipitate was collected and stored. ¹H-NMR confirmed the product (246mg, 49.2%).

Example 14 Synthesis of Ethyl2-([1,1′-biphenyl]-2-ylamino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (319 mg, 1.491mmol) and [1,1′-biphenyl]-2-amine (252 mg, 1.491 mmol) were dissolved in10 mL of ethanol and heated at 80° C. overnight. After heating, thesolution was run on thin layer chromatography and filtered. Theprecipitate was collected and stored. ¹H-NMR confirmed the product.

Example 15 Synthesis of Ethyl6-oxo-2-((4-piperidin-1-yl)amino)-1,6-dihydropyrimidine-5-carboxylate(HGN-0034)

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (313 mg, 1.460mmol) and 4-(piperidin-1yl)aniline (257 mg, 1.460 mmol) were dissolvedin 10 mL of ethanol and heated at 80° C. overnight. After heating, thesolution was run on thin layer chromatography and filtered. Theprecipitate was collected and stored. ¹H-NMR confirmed the product(HGN-0034) (272 mg, 54.4% yield).

Example 16 Synthesis of Ethyl2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxylate

Ethyl sodium 5-(ethoxycarbonyl)-2-(methylthio)-4-oxo-4H-pyrimidin-3-ide(3.31 g, 14.00 mmol) was dissolved in 300 mL of methanol. 2 mL of HClwas added to the solution, which was condensed to yield the product.

Example 17 Synthesis of Ethyl2-((1H-indazol-6-yl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(HGN-0040)

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (358 mg, 1.671mmol) and 1-H-indazol-6-amine (222 mg, 1.671 mmol) were dissolved in 10mL of ethanol and heated at 80° C. overnight. After heating, thesolution was run on thin layer chromatography and filtered. Theprecipitate was collected and stored. ¹H-NMR confirmed the product (227mg, 45.4% yield).

Example 18 Synthesis of Ethyl2-((2,3-dihydro-1H-inden-2-yl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (358 mg, 1.67mmol) and 2,3-dihyrdro-1H-inden-2-amine (222 mg, 1.67 mmol) weredissolved in 10 mL of ethanol and heated at 80° C. overnight.Precipitate was not evident so the solution was heated up to 90° C.overnight. The product was then recovered.

Example 19 Synthesis of2-(4-((5-(ethoxycaronyl)-6-oxo-1,6-dihydropyrimidin-2-yl)amino)phenyl)aceticacid (HGN-0041)

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (338 mg, 1.576mmol) and 2-(4-aminophenyl)acetic acid (238 mg, 1.576 mmol) weredissolved in 10 mL of ethanol and heated at 80° C. overnight. Afterheating, the solution was run on thin layer chromatography and filtered.The precipitate was collected and stored. ¹H-NMR confirmed the product(HGN-0041) (235 mg, 47%).

Example 20 Synthesis of Ethyl2-((1H-indazol-5-yl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(HGN-0042)

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihyropyrimidine-5-carboxylate (358 mg, 1.671mmol) and 1H-indazol-5-amine (222 mg, 1.671 mmol) were dissolved in 10mL of ethanol and heated at 80° C. overnight. After heating, thesolution was run on thin layer chromatography and filtered. Theprecipitate was collected and stored. ¹H-NMR confirmed the product(HGN-0042) (441 mg, 88% yield).

Example 21 Synthesis of Sodium2-((4-morpholinophenyl)amino)-4-oxidopyrimidine-5-carboxylate

The mixture of ethyl2-((4-morpholinophenyl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(210 mg, 0.610 mmol) and sodium hydroxide (1M) were heated at 105° C.for 4 hours followed by heating at 40° C. overnight. Then the solutionwas condensed to recover the final product (290 mg, 132% yield). ¹H-NMRconfirmed the product.

Example 22 Synthesis of Sodium2-([1,1′-biphenyl]-3-ylamino)-4-oxidopyrimidine-5-carboxylate (HGN-0044)

The mixture of ethyl2-(([1,1′-biphenyl]-3-ylamino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(179 mg, 0.534 mmol) and sodium hydroxide (1M) were heated at 105° C.for 4 hours followed by heating at 40° C. overnight. Then the solutionwas condensed to recover the final product. ¹H-NMR confirmed the product(HGN-0044) (170 mg, 91% yield).

Example 23 Synthesis of Sodium4-oxido-2-((piperidin-1-yl)phenyl)amino)pyrimidine-5-carboxylate(HGN-0036)

The mixture of ethyl6-oxo-2-((4-(piperidin-1-yl)phenyl)amino-1,6-dihydropyrimidine-5-carboxylate(219 mg, 0.64 mmol) and sodium hydroxide (1M) were heated at 105° C. for4 hours followed by heating at 40° C. overnight. Then the solution wascondensed to recover the final product. ¹H-NMR confirmed the product(240 mg, 105% yield).

Example 24 Synthesis of Sodium2-((1H-indazol-6-yl)amino)-4-oxoidopyrimidine-5-carboxylate (HGN-0045)

The mixture of ethyl2-((1H-indazol-6-yl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(144 mg, 0.481 mmol) and sodium hydroxide (1M) were heated at 105° C.for 4 hours followed by heating at 40° C. overnight. Then the solutionwas condensed to recover the final product. ¹H-NMR confirmed the product(140 mg, 92% yield).

Example 25 Synthesis of Sodium2-((4-(carboxylatetomethyl)phenyl)amino)-4-oxoidopyrimidine-5-carboxylate

The mixture of2-(4-((5-(ethoxycarbonyl)-6-oxo-1,6-dihyrdropyrimidin-2-yl)amino)phenyl)aceticacid (206 mg, 0.649 mmol) and sodium hydroxide (1M) were heated at 105°C. for 4 hours followed by heating at 40° C. overnight. Then thesolution was condensed to recover the final product. ¹H-NMR confirmedthe product.

Example 26 Synthesis of Sodium2-((1H-indazol-5-yl)amino)-4-oxoidopyrimidine-5-carboxylate (HGN-0046)

The mixture of ethyl2-((1H-indazol-5-yl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(292 mg, 0.976 mmol) and sodium hydroxide (1M) were heated at 105° C.for 4 hours followed by heating at 40° C. overnight. Then the solutionwas condensed to recover the final product. ¹H-NMR confirmed the product(300 mg, 98% yield).

Example 27 Synthesis of Ethyl2-((furan-2-ylmethyl)amino)-6-oxo-1,6-diydropyrimidine-5-carboxylate(HGN-0047)

The mixture of ethyl2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (300 mg, 1.4mmol) and furan-2-ylmethanamine (136 mg, 1.4 mmol) were mixed in ethanoland refluxed overnight. The precipitate was filtered to recover thefinal product. ¹H-NMR confirmed the product (200 mg, 54.3% yield).

Example 28 Synthesis of Ethyl2-((furan-2-ylmethyl)amino)-6-oxo-1,6-diydropyrimidine-5-carboxylic acid

Ethyl2-((furan-2-ylmethyl)amino)-6-oxo-1,6-diydropyrimidine-5-carboxylate(HGN-0047) (150 mg, 0.57 mmol) was suspended in 5 mL of dioxane and then1M NaOH was added dropwise. The reaction mixture was heated under refluxfor 4 hours and at room temperature overnight. The solvent was removedand ¹H-NMR confirmed the product (204 mg, 152% yield).

Example 29 Synthesis of6-oxo-2-(((tetrahydrofuran-2-yl)methyl)amino)-1,6-diydropyrimidine-5-carboxylicacid

Ethyl6-oxo-2-(((tetrahydrofuran-2-yl)methyl)amino)-1,6-diydropyrimidine-5-carboxylate(126 mg, 0.471 mmol) was suspended in 5 mL of dioxane and then 1M NaOHwas added dropwise. The reaction mixture was heated under reflux for 4hours and at room temperature overnight. The solvent was removed and¹H-NMR confirmed the product (191 mg, 169% yield).

Example 30 Synthesis of ethyl6-oxo-2-(phenylamino)-1,6-diydropyrimidine-5-carboxylate

Ethyl 2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (1 g,4.67 mmol) was added into a stirred solution of aniline (522 mg, 5.60mmol). The reaction mixture was heated to 160° C. for 2 hours. Thereaction mixture was cooled down and recrystalized in DMF with water(1:1). The mixture was filtered and titrated the residue with ethanol.¹H-NMR confirmed the product (about 100 mg).

¹H-NMR: δ1.22 (t, 3H, CH₃), δ4.2 (q, 2H, CH₂), δ7.1 (t, 1H, H-Ph C4),δ7.3 (t, 2H, H-Ph C3), δ7.6 (d, 2H, H-Ph C2), δ8.0 (s, 1H, NH), δ8.5 (s,1H, H-pyrimidine)

Example 31 Synthesis of ethyl6-oxo-2-(thiazol-2-ylamino)-1,6-diydropyrimidine-5-carboxylate

Ethyl 2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (1 g,4.67 mmol) was added into a stirred solution of thiazol-2-amine (561 mg,5.60 mmol). The reaction mixture was heated to 160° C. for 2 hours. Thenthe reaction mixture was cooled down and recrystalized in DMF with water(1:1). The mixture was filtered and titrated the residue with ethanol.¹H-NMR confirmed the product (about 300 mg).

¹H-NMR: δ1.35 (t, 3H, H—CH₃), δ4.15 (q, 2H, H—CH₂), δ7.1 (t, 1H,H-thiazol C5), δ7.4 (d, 1H, H-thiazol C4), δ8.48 (s, 1H, H-pyrimidine)

Example 32 Synthesis of6-oxo-2-(phenylamino)-1,6-diydropyrimidine-5-carboxylic acid

Sodium hydroxide (1M) was added to ethyl6-oxo-2-(phenylamino)-1,6-dihydropyrimidine-5-carboxylate (0.1 g, 0.386mmol) in 1 mL methanol and stirred at 80° C. for 2 hours and then 40° C.overnight. Then the reaction mixture was cooled down, and the solventwas removed. The reaction mixture was then diluted with water and the pHwas adjusted to about 3.0. The reaction mixture was filtered, and thesolid was collected and dried. ¹H-NMR confirmed the structure (about 10mg).

¹H-NMR: δ7.1 (t, 1H, H-Ph C4), δ7.3 (t, 2H, H-Ph C3), δ7.6 (d, 2H, H-PhC2), δ8.0 (s, 1H, NH), δ8.5 (s, 1H, H-pyrimidine), δ9.7 (s, 1H, OH).

Example 33 Synthesis of6-oxo-2-(thiazol-2-ylamino)-1,6-diydropyrimidine-5-carboxylic acid

Sodium hydroxide (1M) was added to ethyl6-oxo-2-(thiazol-2-ylamino)-1,6-dihydropyrimidine-5-carboxylate (360 mg,1.352 mmol) in 1 mL methanol and stirred at 80° C. for 2 hours and then40° C. overnight. Then the reaction mixture was cooled down, and thesolvent was removed. The reaction mixture was then diluted with waterand the pH was adjusted to about 3.0. The reaction mixture was filtered,and the solid was collected and dried. ¹H-NMR confirmed the structure(about 20 mg).

¹H-NMR: δ7.1 (d, 1H, H-thiazol C5), δ7.4 (d, 1H, H-thiazol C4), δ8.48(s, 1H, H-pyrimidine), δ9.5 (s, 1H, OH).

Example 34 Synthesis of2-4-bromophenyl)amino)-6-oxo-1,6-diydropyrimidine-5-carboxylic acid

Sodium hydroxide (1M) was added to ethyl2-((4-bromophenyl)amino)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (155mg, 0.458 mmol) in 2 mL dioxane and hydrolyzed at 80° C. for 2 hours andthen 40° C. overnight. Then the reaction mixture was cooled down, andthe dioxane was removed. The reaction mixture was then acidified with INHCl so the pH was adjusted to about 3.0. The reaction mixture wasfiltered, and the solid was collected and dried. ¹H-NMR confirmed thestructure.

Example 35 IspF Binding Inhibition

Binding affinities between IspF and compounds synthesized herein weremeasured by surface plasmon resonance (SPR).

Methods

The binding constants of synthesized inhibitors were analyzed usingsurface plasmon resonance (SPR). All SPR experiments were performedusing a Reichert SR7500DC instrument (Reichert, N.Y, USA) withcarboxymethyl dextran hydrogel surface sensor chips (500,000 Daltonmolecular weight carboxymethyl dextran chips, Reichert, N.Y, USA). IspFwas immobilized using amine-coupling chemistry. The sensor chip surfacewas activated with a 10 min injection of a mixture of 11.5 mg/mLN-hydroxysucciniminde and 76.5 mg/mL 1-ethyl-3-(3-dimethylamino propyl)carbodiimide hydrochloride at a flow rate of 20 μl/min. IspF was dilutedto final concentration of 60 μg/ml with 5 mM acetate buffer, pH 5.0 andimmobilized with a 10 min injection over the left channel at a flow rateof 20 μl/min. Typically, 3000 μRIU-4000 μRIU of IspF was immobilized. Toquench excess succinaminde ester groups, 1M Ethanolamine, pH 8.5 wasinjected for 4 min at a 50 μl/min flow rate. All SPR experiments wereperformed at 25° C. using PBS-D (50 mM sodium phospate/pH 7.4, 150 mMsodium chloride, and 5% DMSO) as a running buffer at a 50 μl/min flowrate. Inhibitors were initially dissolved in DMSO. Samples were dilutedsuch that 5% DMSO was maintained in all samples. A concentration seriesof inhibitor, as well as periodic buffer injections, were individuallyinjected and collected for approximately 5 minutes. Data were analyzedusing Scrubber2 (Biologic Software).

Results

The binding affinity between IspF inhibitor compounds and IspF weremeasured by SPR. The results can be used as a screening tool to indicatewhich compounds should be evaluated further. The results are provided interms of the dissociation constant (K_(d)) between IspF and theinhibitor compounds. No binding indicated a lack of inhibition. Lowerconcentrations indicated a higher degree of inhibition.

TABLE 1 BINDING AFFINITY IspF INHIBITORS AND IspF Inhibitor K_(d)HGN-0001 no binding HGN-0002 not soluble HGN-0003  82 uM HGN-0005 176 uMHGN-0013 non-specific HGN-0018 no binding HGN-0019 380 uM HGN-0020 204uM HGN-0024 784 uM HGN-0025 no binding HGN-0026 no binding HGN-0027 nobinding HGN-0028 305 uM HGN-0030 178 uM HGN-0035 not soluble HGN-00037not soluble HGN-0038 not soluble HGN-0039 non specific HGN-0040 nobinding HGN-0041 not soluble HGN-0042 not soluble HGN-0045 non specificHGN-0046 non specific HGN-0048 486 uM HGN-0052 no binding HGN-0055 nobinding HGN-0056 no binding HGN-0060 not soluble HGN-0061 no bindingHGN-0062 no binding HGN-0063 10.7 mM 

Example 36 Growth Inhibition of Burkholderia Spp. by IspF InhibitorCompounds

Growth inhibition properties of compounds HGN-0023, HGN-0027, HGN-0028,and HGN-0029 against B. thailandensis E264 and B. pseudomallei 1026b andBp340 were evaluated.

Methods

Overnight cultures of Burkholderia strains (obtained from HerbertSchweizer at the University of Colorado) were grown in low-salt LB(LSLB; 10 g/L tryptone, 5 g/L yeast extract, 1.25 g/L NaCl) at 37° C.The Burkholderia strains were diluted to 0.2 OD₆₀₀ in LSLB. Stocks ofHGN compounds (40 mM in DMSO, except HGN-0028 at 20 mM) were diluted to1 mM, then two-fold dilutions (1 mL each) in LSLB (500 μM, 250 μM, 125μM, 62.5 μM, 31.25 μM) were prepared. In a 96-well microtiter plate, 160μL of the inhibitor in LSLB at each concentration were added to thewells in triplicate. Then 40 μL of the 0.2 OD₆₀₀ bacterial suspension towas added each well (final OD₆₀₀=0.04, total well volume=200 μL). Ascontrols, 40 μL of LSLB, DMSO, or kanamycin were added (finalconcentration 300 μg/mL) to wells in triplicate. The plates wereincubated for 20 h at 37° C. with shaking in an Infinite M200 platereader (Tecan Group Ltd., Männedorf, Switzerland). The plate was read at15 min. intervals at 600 nm wavelength. The OD₆₀₀ values were graphedover time (FIGS. 1-6).

Results

Initial results (FIG. 1) indicated several candidate compounds tofurther evaluate inhibition of the growth of B. pseudomallei. From FIG.2, tests indicated that HGN-0027 and HGN-0028 inhibited the growth of B.pseudomallei over time. Further study tested the concentration effectsof HGN-0023, -0027, -0028, and -0029 on the the growth of B.pseudomallei over time. These studies confirmed that HGN-0023 (FIG. 3)and HGN-0029 (FIG. 6) did not inhibit the growth of B. pseudomallei.Whereas, HGN-0027 was a potent inhibitor, at multiple concentrations, ofthe growth of B. pseudomallei (FIGS. 4, 7, and 8). HGN-0028 was dosedependent in its ability to inhibit B. pseudomallei growth (FIGS. 5, 9.and 10). Lower concentrations of HGN-0028 did not inhibit B.pseudomallei growth.

Example 37

The compounds can be grouped by chemical class, Pyrimidine IspFinhibitors (Table 2), Imidazole IspF inhibitors (Table 3), Fusion IspFinhibitors (Table 4), and miscellaneous IspF inhibitors (Table 5). Thetables below also summarize the bacterial inhibition of the compounds.

TABLE 2 Pyrimidine IspF inhibitors % Growth Inhibition^(a) ChemicalEndpoint Growth Designation Formula Structure (24 h) Curve HGN-0017C₁₂H₁₇N₃O₄

ND 0.00^(b) HGN-0047 C₁₂H₁₃N₃O₄

0.00^(b) ND HGN-0041 C₁₅H₁₅N₃O₅

0.00^(b) ND HGN-0121 C₁₄H₁₄ClN₃O₃

0.00^(b) ND HGN-0123 C₁₄H₁₄ClN₃O₃

0.00^(b) ND HGN-0125 C₁₄H₁₄ClN₃O₃

 23.89 ± 36.14 ND HGN-0038 C₁₅H₁₆ClN₃O₃

0.00^(b) ND HGN-0119 C₁₅H₁₆ClN₃O₃

0.00^(b) ND HGN-0127 C₁₄H₁₃C₁₂N₃O₃

 3.47 ± 2.20 ND HGN-0129 C₁₄H₁₃Cl₂N₃O₃

64.42 ± 2.77 ND HGN-0131 C₁₅H₁₅Cl₂N₃O₃

0.00^(b) ND HGN-0117 C₁₅H₁₅C₁₂N₃O₃

0.00^(b) ND HGN-0135 C15H15Cl2N3O3

0.00^(b) ND HGN-0133 C₁₄H₁₄FN₃O₃

0.00^(b) ND HGN-0113 C₁₄H₁₄FN₃O₃

0.00^(b) ND HGN-0111 C₁₅H₁₆FN₃O₃

 31.31 ± 39.32 ND HGN-0115 C₁₅H₁₆FN₃O₃

0.00^(b) ND HGN-0016 C₁₃H₁₁ClN₄O₄

ND ND HGN-0040 C₁₄H₁₃N₅O₃

16.39 ± 6.29 ND HGN-0042 C₁₄H₁₃N₅O₃

20.70 ± 0.71 ND HGN-0034 C₁₈H₂₂N₄O₃

86.20 ± 0.16 ND HGN-0037 C₁₉H₁₇N₃O₃

L^(c) ND HGN-0033 C₁₉H₁₇N₃O₃

 5.07 ± 2.01 ND HGN-0039 C₁₉H₁₇N₃O₃

0.00^(b) ND HGN-0015 C₁₈H₁₆N₄O₃

ND 14.43 ± 2.38  HGN-0012 C₈H₆N₄O₃S

ND ND HGN-0077 C₁₆H₁₂N₄O₃

ND ND HGN-0079 C₁₁H₈BrN₃O₃

ND ND HGN-0011 C₁₁H₉N₃O₃

ND ND HGN-0035 C₁₇H₁₃N₃O₃

91.14 ± 1.18 ND HGN-0050 C₁₀H₁₁N₃Na₂O₄

32.99 ± 9.22 ND HGN-0049 C₁₀H₇N₃Na₂O₄

12.25 ± 0.40 ND HGN-0126 C₁₂H₈ClN₃Na₂O₃

34.34 ± 3.24 ND HGN-0124 C₁₂H₈ClN₃Na₂O₃

30.67 ± 2.67 ND HGN-0122 C₁₂H₈ClN₃Na₂O₃

23.02 ± 8.79 ND HGN-0028 C₁₃H₁₀ClN₃Na₂O₃

ND 68.20 ± 2.12  HGN-0120 C₁₃H₁₀ClN₃Na₂O₃

80.30 ± 0.40 ND HGN-0118 C₁₃H₉Cl₂N₃Na₂O₃

94.50 ± 4.42 ND HGN-0128 C₁₂H₇Cl₂N₃Na₂O₃

37.46 ± 0.21 ND HGN-0130 C₁₂H₇Cl₂N₃Na₂O₃

45.59 ± 2.35 ND HGN-0132 C₁₃H₉Cl₂N₃Na₂O₃

 72.24 ± 11.83 ND HGN-0136 C₁₃H₉Cl₂N₃Na₂O₃

16.71 ± 5.04 ND HGN-0134 C₁₂H₈FN₃Na₂O₃

26.05 ± 6.37 ND HGN-0114 C₁₂H₈FN₃Na₂O₃

 5.94 ± 5.46 ND HGN-0112 C₁₃H₁₀FN₃Na₂O₃

 19.82 ± 16.53 ND HGN-0116 C₁₃H₁₀FN₃Na₂O₃

20.46 ± 3.47 ND HGN-0014 C₁₁H₆BrN₃Na₂O₃

15.52 ± 0.41 ND HGN-0045 C₁₂H₇N₅Na₂O₃

0.00^(b) ND HGN-0046 C₁₂H₇N₅Na₂O₃

15.12 ± 4.25 ND HGN-0036 C₁₆H₁₆N₄Na₂O₃

 22.38 ± 21.24 ND HGN-0013 C₁₆H₁₀N₄Na₂O₃

ND 6.26 ± 1.00 HGN-0044 C₁₇H₁₁N₃Na₂O₃

 1.48 ± 7.90 ND ^(a)Growth inhibition calculated relative to DMSOcontrol ^(b)Growth with inhibitor was equal to or greater than DMSOcontrol ^(c)Limited solubility prevented accurate determination ofgrowth ND, not done

TABLE 3 Imidazole IspF inhibitors. % Growth Inhibition^(a) ChemicalEndpoint Growth Designation Formula Structure (24 h) Curve HGN-0029 andHGN-0056 C₉H₈N₂O

ND 23.58 ± 0.54  HGN-0018 C₁₂H₁₄N₂O

ND ND HGN-0019 C₁₄H₁₂N₂O₂

ND ND HGN-0020 C₁₉H₁₈ClN₃O

ND 1.26 ± 2.33 HGN-0137 C₂₀H₁₉N₃O₂

0.00^(b) ND HGN-0021 C16H14N2O

ND ND HGN-0022 and HGN-0052 (HCl salt) C₁₇H₁₆N₂O

0.00^(b) ND HGN-0048 C₁₇H₁₇ClN₂O₂

ND ND HGN-0091 C₁₆H₁₃ClN₂O

L^(c) ND HGN-0090 C₁₆H₁₃ClN₂O

L^(c) ND HGN-0094 C₁₆H₁₃ClN₂O

L^(c) ND HGN-0108 C₁₇H₁₅ClN₂O

L^(c) ND HGN-0051 C₁₇H₁₅ClN₂O

0.00^(b) ND HGN-0096 C₁₇H₁₅ClN₂O

L^(c) ND HGN-0092 C₁₆H₁₂Cl₂N₂O

25.41 ± 2.79  ND HGN0098 C₁₇H₁₄Cl₂N₂O

0.00^(b) ND HGN-0099 C₁₇H₁₄Cl₂N₂O

0.00^(b) ND HGN-0100 C₁₇H₁₄Cl₂N₂O

L^(c) ND HGN-0109 C₁₆H₁₃FN₂O

24.77 ± 10.79 ND HGN-0106 C₁₆H₁₃FN₂O

33.79 ± 16.09 ND HGN-0093 C₁₆H₁₃FN₂O

58.12 ± 2.17  ND HGN-0095 C₁₇H₁₅FN₂O

88.27 ± 0.48  ND HGN-0097 C₁₇H₁₅FN₂O

 4.27 ± 14.54 ND HGN-0107 C₁₇H₁₅FN₂O

2.75 ± 2.62 ND ^(a)Growth inhibition calculated relative to DMSO control^(b)Growth with inhibitor was equal to or greater than DMSO control^(c)Limited solubility prevented accurate determination of growth ND,not done

TABLE 4 Fusion IspF inhibitors. % Growth Inhibition^(a) ChemicalEndpoint Growth Designation Formula Structure (24 h) Curve HGN-0005C₁₅H₁₇N₅O₅

ND 0.00^(b) HGN-0006 C₁₅H₁₆N₆O₅S

0.00^(b) ND HGN-0007 C₂₀H₁₉F₆N₇O₉

0.00^(b) ND HGN-0008 C₁₇H₁₉F₆N₅O₁₀S

0.00^(b) ND HGN-0102 C₁₆H₁₈N₆O₅S

0.00^(b) ND ^(a)Growth inhibition calculated relative to DMSO control^(b)Growth with inhibitor was equal to or greater than DMSO control ND,not done

TABLE 5 Miscellaneous IspF inhibitors. % Growth Inhibition^(a) ChemicalEndpoint Growth Designation Formula Structure (24 h) Curve HGN-0031C₁₁H₁₂N₂O

ND 0.00^(b) HGN-0061 C₁₂H₁₄N₂O

ND ND HGN-0063 C₁₇H₁₆N₂O

ND ND HGN-0060 C₂₃H₂₀N₂O

ND ND HGN-0062 C₁₈H₁₈N₂O

ND ND HGN-0078 C₁₀H₉N₅

ND ND HGN-0080 C₁₀H₁₁N₅O

ND ND HGN-0004 C₈H₇N₅O₂S

0.00^(b) ND HGN-0002 C₁₂H₁₀N₆O₂

ND ND HGN-0010 C₁₀H₈N₆O₂S

7.22 ± 4.24 ND HGN-0009 C₁₁H₁₀N₆O₂S

0.00^(b) ND ^(a)Growth inhibition calculated relative to DMSO control^(b)Growth with inhibitor was equal to or greater than DMSO control ND,not done

Example 38 Growth Inhibition of Malaria by IspF Inhibitor Compounds

The compounds described herein were also tested for their ability toinhibit the growth of the causative agent of malaria, Plasmodiumfalciparum, a protozoan parasite.

Methods

SYBR assay: The method of testing inhibition has been previouslydescribed (Co et al., 2009). Briefly, D6, C235 and W2 P. falciparumstrains were maintained in continuous long-term cultures in tissueculture medium. Cultures and assays were grown and conducted at 37° C.under a humidified atmosphere of 5% CO₂ and 5% O₂, with a balance of N₂using either tissue culture medium or folic acid-free medium. Parasitesat 1% parasitemia and 2% hematocrit were added to predosed 96-wellplates and incubated for 72 h. Lysis buffer (20 mM Tris HCl, 5 mM EDTA,0.008% saponin, and 0.08% Triton®-X with SYBR® green I dye wassubsequently added to the plates and incubated for 1 h at ambient roomtemperature. Fluorescence was determined, and the plates were examinedfor relative fluorescence units (RFUs) per well. The drug concentrationswere transformed and the data were then analyzed with Prism to yielddrug 50% inhibitory concentrations (IC50s). Green D6, Green C235, andGreen W2 are three strains in P. falciparum. W2 is chloroquine resistantand mefloquine sensitive, D6 is chloroquine sensitive but naturally lesssusceptible to mefloquine, C235 is resistant to mefloquine, chloroquine,and pyrimethamine.

Results

The data are provided in Tables 6-9 below.

TABLE 6 Anti-malarial data for pyrimidine IspF inhibitors. SYBR SYBRSYBR Green D6 Green C235 Green W2 Designation IC₅₀ (uM) IC₅₀ (uM) IC₅₀(uM) HGN-0017 >40 >40 >40 HGN-0047 >40 >40 >40 HGN-0041 >40 >40 >40HGN-0121 34.52 >40 >40 HGN-0123 ND ND ND HGN-0125 >40 19.68 16HGN-0038 >40 >40 >40 HGN-0119 ND ND ND HGN-0127 >40 >40 17.7 HGN-012926.95 32.98 13.19 HGN-0131 ND ND ND HGN-0117 ND ND ND HGN-0135 ND ND NDHGN-0133 >40 >40 >40 HGN-0113 29.29 >40 >40 HGN-0111 >40 >40 >40HGN-0115 17.45 27.58 17.59 HGN-0016 ND ND ND HGN-0040 >40 >40 >40HGN-0042 >40 >40 >40 HGN-0034 >40 >40 >40 HGN-0037 >40 >40 >40HGN-0033 >40 >40 >40 HGN-0039 >40 >40 >40 HGN-0015 >40 >40 >40 HGN-0012ND ND ND HGN-0077 ND ND ND HGN-0079 ND ND ND HGN-0011 ND ND ND HGN-0035ND ND ND HGN-0050 ND ND ND HGN-0049 0.55 0.74 0.95 HGN-0126 >40 >40 >40HGN-0124 ND ND ND HGN-0122 ND ND ND HGN-0028 >40 >40 >40 HGN-0120 ND NDND HGN-0118 ND ND ND HGN-0128 ND ND ND HGN-0130 ND ND ND HGN-0132 ND NDND HGN-0136 ND ND ND HGN-0134 ND ND ND HGN-0114 ND ND ND HGN-0112 ND NDND HGN-0116 ND ND ND HGN-0014 >40 >40 >40 HGN-0045 >20 >20 >20 HGN-0046ND ND ND HGN-0036 ND ND ND HGN-0013 >40 >40 >40 HGN-0044 >40 >40 >40

TABLE 7 Anti-malarial data for imidazole IspF inhibitors. SYBR SYBR SYBRGreen D6 Green C235 Green W2 Designation IC₅₀ (uM) IC₅₀ (uM) IC₅₀ (uM)HGN-0029 >40 >40 >40 and HGN-0056 HGN-0018 >40 >40 >40 HGN-0019 11.313.3 13.6 HGN-0020 4.2 7.2 5.6 HGN-0137 HGN-0021 6.0 7.7 6.0HGN-0022 >40 >40 >40 and HGN-0052 (HCl salt) HGN-0048 >40 >40 >40HGN-0091 3.175 4.874 3.526 HGN-0090 5.797 6.153 3.943 HGN-0094 8.8769.116 6.221 HGN-0108 11.48 13.81 7.219 HGN-0051 1.7 3.4 0.59 HGN-00969.798 12.92 7.898 HGN-0092 ND ND ND HGN0098 10.16 14.38 8.381 HGN-00995.362 5.436 4.251 HGN-0100 4.275 9.366 5.647 HGN-0109 4.798 5.735 3.163HGN-0106 3.001 3.524 4.099 HGN-0093 5.829 6.345 3.36 HGN-0095 6.1 6.5944.048 HGN-0097 10.16 5.178 2.12 HGN-0107 5.653 7.291 4.088

TABLE 8 Anti-malarial data for fusion series IspF inhibitors. SYBR SYBRSYBR Green D6 Green C235 Green W2 Designation IC₅₀ (uM) IC₅₀ (uM) IC₅₀(uM) HGN-0005 >40 >40 >40 HGN-0006 ND ND ND HGN-0007 ND ND ND HGN-0008ND ND ND HGN-0102 >40 >40 >40

TABLE 9 Anti-malarial data for miscellaneous IspF inhibitors. SYBR SYBRSYBR Green D6 Green C235 Green W2 Designation IC₅₀ (uM) IC₅₀ (uM) IC₅₀(uM) HGN-0068 8.374 6.119 5.988 HGN-0024 >40 >40 >40 HGN-002312.9 >40 >40 HGN-0082 >40 >40 >40 and HGN-0101 HGN-0054 28.9 >40 11.5HGN-0031 >40 >40 >40 HGN-0061 6.1 8.3 6.4 HGN-0063 >40 >40 >40 HGN-00601.6 2.7 3.1 HGN-0062 3.8 8.3 7.3 HGN-0078 HGN-0080 HGN-0004 >40 >40 >40HGN-0002 >40 >40 >40 HGN-0010 >40 >40 >40 HGN-0009 >40 >40 >40HGN-0085 >40 >40 >40 HGN-0083 8.49 12.03 10.4 HGN-0084 >40 >40 >40HGN-0055 18.8 >40 19 and HGN-0089 HGN-0087 >40 >40 >40HGN-0088 >40 >40 >40 HGN-0025 >40 >40 >40 HGN-0026 >40 >40 >40 HGN-00577.6 22.8 15.0 HGN-0058 >40 >40 >40 HGN-0059 >40 >40 >40 HGN-0030 >40 >40>40

PUBLICATIONS

All publications cited in this application are herein incorporated byreference.

-   -   Begley et al. (2010) Chem. Biol. Drug Des. 76(3):218-233.    -   Brown, A. C., and Parish, T. (2008) BMC Microbiol. 8: 78.    -   Co et al. (2009) Antimicrob. Agents Chemother. 53:2557-2563.    -   Gershenzon, J., and Dudareva, N. (2007) Nat. Chem. Biol. 3:        408-414.    -   Hunter, W. N. (2007) J. Biol. Chem. 282; 21573-21577.    -   Kuzuyama, T. et al. (1999) Biosci., Biotechnol., Biochem. 63:        776-778.    -   Mayer et al. (1999) Angew Chem. Int. Ed. 38(12):1784-1788.    -   Piotto et al. (1992) J. Biomol. NMR 2(6):661-665.    -   Zhang, B. et al. (2011) Biochemistry 50: 3570-3577.

The invention claimed is:
 1. A method of treating a bacterial infectionin a mammal, the method comprising administering to the mammal atherapeutically effective amount of the compound of Formula I or FormulaII

wherein R¹ is OH or OR⁵, wherein R⁵ is lower alkyl, acyl, or CH₂OCOCH₃;R² is alkyl, alkylaryl, arylheteroaryl, or heteroaryl; R³ is H or loweralkyl; and R⁴ is H or lower alkyl, or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, further comprising administering atleast one other antimicrobial agent.
 3. A method of treating a protozoaninfection in a mammal, the method comprising administering to the mammala therapeutically effective amount of the compound of Formula I orFormula II

wherein R¹ is OH or OR⁵, wherein R⁵ is lower alkyl, acyl, or CH₂OCOCH₃;R² is alkyl, alkylaryl, arylheteroaryl, or heteroaryl; R³ is H or loweralkyl; and R⁴ is H or lower alkyl, or a pharmaceutically acceptable saltthereof.
 4. A method for the treatment of bacterial, protozoan, andparasitic infections, the method comprising: (a) obtaining apharmaceutical composition comprising a therapeutically effective amountof the compound of Formula I or Formula II and a pharmaceuticallyacceptable carrier

 wherein R¹ is OH or OR⁵, wherein R⁵ is lower alkyl, acyl, or CH₂OCOCH₃;R² is alkyl, alkylaryl, arylheteroaryl, or heteroaryl; R³ is H or loweralkyl; and R⁴ is H or lower alkyl, or a pharmaceutically acceptable saltthereof; and (b) administering the composition to a mammal in needthereof.